Overcoming AlbD Protease Resistance and Improving Potency: Synthesis and Bioactivity of Antibacterial Albicidin Analogues with Amide Bond Isosteres

Article abstract of DOI:10.1021/acs.orglett.1c02312

Albicidin is a potent antibacterial oligoaromatic peptide that is susceptible to the protease AlbD, a resistance factor. This potentially restricts the use of albicidin as a drug. To overcome this obstacle, we synthesized and evaluated six analogues with

Full text of DOI:10.1021/acs.orglett.1c02312

pubs.acs.org/OrgLett
Letter
Overcoming AlbD Protease Resistance and Improving Potency:
Synthesis and Bioactivity of Antibacterial Albicidin Analogues with
Amide Bond Isosteres
Leonardo Kleebauer, Lieby Zborovsky, Kay Hommernick, Maria Seidel, John B. Weston,
and Roderich D. Sussmuth*
̈
Cite This: https://doi.org/10.1021/acs.orglett.1c02312
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Albicidin is a potent antibacterial oligoaromatic
peptide that is susceptible to the protease AlbD, a resistance factor.
This potentially restricts the use of albicidin as a drug. To overcome
this obstacle, we synthesized and evaluated six analogues with
isosteric replacement of the key amide link. Protease stability was
established while maintaining the antibacterial activity, including
three analogues with up to eight times higher activity compared with
the natural albicidin.
he development of antibacterial resistance toward
Tmedically used antibiotics is responsible for the emergence
of a life-threatening health crisis. At the same time, big
pharmaceutical companies are dropping out of antimicrobial
research due to its low profitability.1 Thus there is an urgent
need for novel highly potent, antibiotic therapeutics.2 The
natural product albicidin produced by Xanthomonas albilineans
was identified but not characterized in 1985.3 It is an
oligoaromatic peptide (Figure 1) that shows remarkable
bioactivity against not only G+ve but also G−ve pathogens,
with bacterial DNA gyrase as a target. Both the structure
elucidation4 and the first total synthesis were accomplished by
our group.5 This led to the reporting of a number of albicidin
structure−activity relationship (SAR) studies describing build-
ing block replacements.6,7
Figure 2. Lead structure (2) and targeted D−E isosteres (3−8).
structure (Figure 1). It consists of six buildings blocks (A:
methylcoumaric acid (MCA), B: para-aminobenzoic acid
(pABA1), C: L-cyanoalanine (Cya), D: para-aminobenzoic
acid (pABA2), and E and F: para-amino-2-hydroxy-3-methox-
ybenzoic acid (pHMBA1 and pHMBA2)) which are linked by
five peptide bonds. Our lead structure aza-His albicidin (2) with
the replacement of Cya by aza-Histidine has an optimized
antibacterial profile and a higher chemical stability (Figure 1).
Albicidin (1) is naturally synthesized according to a
nonribosomal biosynthesis mechanism and has an unusual
Received: July 10, 2021
Figure 1. Structure of albicidin (1) and aza-His albicidin (2) and the
assignment of building blocks A−F.
© XXXX The Authors. Published by
https://doi.org/10.1021/acs.orglett.1c02312
American Chemical Society
Org. Lett. XXXX, XXX, XXX−XXX
A

Organic Letters
pubs.acs.org/OrgLett
Letter
Scheme 1. Synthesis of the Key Building Block 12 for the
Synthesis of Albicidin Analogues 3−7
Scheme 3. Synthesis of the D−E−F Fragments 28d−f
Scheme 2. Palladium-Mediated Cross-Couplings as the Key
Steps toward the D−E−F Fragments (28a and 28b)
overall conformational requirements of the albicidin structure
(Figure 1, 1). Hence, ethane (3), ethene (4), and ethyne (5)
linkages connecting building blocks D and E were chosen to
investigate the bond flexibility or rotatability. The (Z)-
fluoroalkene (7) seems particularly attractive, as the introduc-
tion of the fluorine provides geometrical similarities to an amide
link. For example, for an amide, the CO bond is 1.23 Å and the
C−N bond is 1.37 Å, and the C−F bond is 1.38 Å and the CC
bond is 1.33 Å.9 Furthermore, the fluorine conserves the
electron-withdrawing effect of the oxygen of the amide link and
polarizes the group to provide weak H-bond acceptor properties
similar to those of the amide bond with a dipole moment of 1.4
D (3.6 D in case of an amide).10 Derivatives (6, 8) (Figure 2) are
more unusual analogues that limit the conformational flexibility
by ring formation.11 An additional challenge was that the
synthesis strategy had to be individually adapted to the synthesis
of each of the analogues. We chose a strategy beginning with the
preparation of the individual D−E isosteric fragments.
The best and most flexible synthetic strategy for amide bond
isosteres 3−5 and 7 (Figure 2) seemed to be the establishment
of the isosteric bond from the same preformed E building block
followed by coupling to different D building blocks rather than
the converse and keeping the D building block constant. Thus
the synthesis of 4-iodo-3-methoxy-2-hydroxybenzoic acid
(Scheme 1, 12) for building block E was achieved in six steps,
starting from o-vanillin (Scheme 1). The starting material o-
vanillin 9 was acetylated, allowing a more selective nitration at
the para position. A Pinnick oxidation followed to give
carboxylic acid (10). Cleavage of the acetyl group and reduction
with palladium on activated charcoal afforded 4-amino-3-
methoxy-2-hydroxy benzoic acid (11). The key diazotisation
step was followed by iodination to provide the tetrasubstituted
4-iodo-3-methoxy-2-hydroxybenzoic acid (12) in good yield.
A number of albicidin resistance factors have been
characterized. These include the endopeptidase AlbD from
Pantoea dispersa.8 The peptidase specifically cleaves between the
D and E building blocks, leading to a complete loss of activity
(Figure 1). Structural optimization of albicidin derivatives is one
way to overcome these resistance factors. Previously, we
synthesized four analogues with isosteric D−E amide replace-
ments (triazole, N-methyl amide, urea, and sulfonamide) and
tested them against AlbD. Cleavage induced by AlbD could be
prevented in all cases (except for the N-methylated amide) but
mostly at the expense of antibiotic activity measured by minimal
inhibitory concentration (MIC).6 Only the triazole isostere
showed an acceptable antibacterial performance (MIC was
decreased by approximately two- to four-fold). To prevent
cleavage of the amide bond located between building blocks D
and E by AlbD and ideally to retain or even to increase the
potency, new isosteric replacements were needed. The challenge
was to identify new isosteres, either mimicking the amide bond
more accurately with regard to steric and electronic properties or
abandoning unnecessary structural features while maintaining
the antibacterial activity.
We carried out the synthesis of six derivatives with amide
bond replacements between building blocks D and E. The
critical feature was the stability toward chemical or enzymatic
hydrolysis, thus disfavoring esters while maintaining the steric
requirement of the peptide motif as much as possible and the
B
https://doi.org/10.1021/acs.orglett.1c02312
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Scheme 4. Synthesis of Albicidin Isosteres (3−8) from D−E−F Fragments (28a−f)
Table 1. Inhibition of E. coli DNA Gyrase, Antibacterial Activity, and Activity against E. coli DSM 1116 in the Presence of AlbD of
a
Synthetic Isosteres 3−8
a
Reference compounds 2, 34, and 35 are marked with *. MIC values are given in μg/mL: ≤0.031, dark green; 0.063−0.250, light green; 0.5−1,
yellow; 2−4, light red; ≥8, red. Activity in the presence of AlbD: ×, inactive; √, active. Gyrase inhibition at a constant concentration of 45 nM of
compounds 1 and 2−8: ↑, for stronger inhibition; ≈, for equal inhibition; ↓, for diminished inhibition compared with albicidin 1.6
To prepare albicidins 3 (alkane) and 4 (alkene), respectively,
4-nitro styrene (Scheme 2, 13) was reacted with the unprotected
tetrasubstituted 2-hydroxy-4-iodo-3-methoxybenzoic acid (12)
in a trans-selective Heck reaction. Protecting groups for the free
phenol and carboxylic acid (benzyl, tert-butyl, MOM, or p-
methoxybenzyl (PMB)) were not suitable for reasons of the
required stability and orthogonality. Finally, we used allyl
protecting groups that could be used only after the Heck
reaction to avoid cross-coupling with the allyl groups. After
protection of both the hydroxy group and the carboxylic acid,
the acid was then deprotected with LiOH in tetrahydrofuran
(THF)/water to give 14.
For the peptide coupling of the D−E fragments with the F
fragment 15, mild reaction conditions were necessary to avoid
C
https://doi.org/10.1021/acs.orglett.1c02312
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
isomerization of the CC double bond. Therefore, coupling
was performed with oxalyl chloride, and the resulting
pseudotripeptide was reduced with zinc powder to afford aniline
28a.
increased activity (up to 4-fold) against G+ve B. subtilis and
Mycobacterium phlei, with only the more polar benzimidazole
isostere 8 (HPLC RT = 6.58 min, Table S1) showing lower
activity (up to eight-fold). The inhibition of the E. coli gyrase of
4, 5, and 7 is stronger than that for albicidin (1) and correlates
with the observed antibacterial activity (Table S1 Figure S2,
Supporting Information).
Next, we focused on the D−E−F fragment of the alkyne 5
(Scheme 2, II). A Pd−Cu-catalyzed Sonogashira coupling
instead of the Heck reaction allowed us to access the D−E
alkyne fragment 17. Using the same protection/deprotection
and coupling steps as those for 28a afforded alkyne 28b.
For the albicidin alkane isostere 3, benzyl instead of allyl
protecting groups were used. This facilitated a one-step
hydrogenation of the alkene (Scheme 4, 30c) to give the alkane
together with concurrent deprotection of the benzyl groups to
give the C−D−E−F fragment (Scheme 4, 31c).
Fluoroalkene 7 was then prepared. Compound 20 (Scheme 3,
I) was synthesized from 1-iodo-4-nitrobenzene (18) and (1-
fluorovinyl)(methyl)diphenylsilane (19) by a cesium-fluoride-
assisted Cu−Pd-catalyzed reaction.12
Regarding the structural features of the isosteres, it appears
that fused ring analogs17 restricting the conformational flexibility
are unfavorable. Furthermore, the D−E link consisting of highly
conformationally flexible sp3 carbons (3) shows that very high
conformational flexibility is not preferable. However, both, the
(Z)-fluoroalkene (7) and the alkyne derivative (5) showed an
enhanced activity compared with the parent structure (2). The
(E)-alkene (4) was less active than the (Z)-fluoroalkene (7),
probably because the (Z)-fluoroalkene is more similar to an
amide (see previous discussion). Remarkably, alkyne 5 is a good
isostere of an amide bond.18 Apparently, the linearity and
stiffness of the D−E moiety due to the sp-hybridized carbons of
5 (180° bond angle) permits good bioactivity, despite a poorer
overlay with the parent compound 2. Another advantage of the
alkyne structure could be the impossibility for cis−trans
isomerization, observable for alkenes, as shown for coumaric
acid (building block A), which is detrimental for antibacterial
activity.6,17
In summary, we have shown that replacement of the D−E
amide bond of albicidin resists protease AlbD cleavage, and at
the same time, potency against both G+ve and G−ve bacteria
can be increased. Our study on amide bond replacements
encourages the application of these isosteres to replace not only
other amides of albicidin or related cystobactamide struc-
tures19−21 but also other peptide structures or aromatic
oligoamides.22,23 Whereas the (Z)-fluoroalkene (7) is a state-
of-the-art well-known isostere,24 to our knowledge, we report
one of the very few examples7 of an alkyne (5) considered to act
as an amide isostere.9 This Letter is a further important step in
understanding the SARs of albicidin. With the compounds 5 and
7, we report two albicidin candidates for future preclinical
evaluation on the path to resolve the antibiotic resistance crises.
Next, we synthesized the cyclic isosteres. The benzimidazole
tripeptide mimic 28e was prepared from nitrobenzaldehyde 22
(Scheme 3, II) and dianiline 23 by annulation followed by
reduction of the nitro group. For the benzofuran cyclic isostere, a
ring fusion to the D fragment (Scheme 3, III, 28f), we prepared
the aryl iodide dipeptide 25 in a nine-step synthesis. (See the SI.)
(Trimethylsilanyl)-acetylene (24) reacted with dipeptide 25
(Scheme 4) by Sonogashira cross-coupling to provide acetylene
26 after a trimethylsilyl (TMS) deprotection with tetra-n-
butylammonium fluoride (TBAF). The Cu−Pd-catalyzed
reaction of iodophenol 27 with 26 has been previously
described.13 After the reduction, we obtained aminobenzofuran
28f.
The final five steps were similar for all six D−E−F fragments
(Scheme 4, 28a−f) and were carried out as follows: N-
Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a cou-
pling agent known for its low rates of racemization14,15 and
successful application in a former albicidin synthesis,16 was used
to couple the D−E−F fragment to the enantiopure C building
block (29). This was followed by a deprotection of first, the allyl
groups (benzyl groups for 30c and 30f) and second, the Boc
group. Employing a strategy we had used previously, the AB-
PCP ester 32 (Scheme 4) was used for the late-stage coupling
step. Finally, the pivaloyloxymethyl (POM) protecting group on
the C fragment was cleaved under basic conditions. This route
worked for five of the six derivatives. When we tried to couple
31f to the AB-PCP (32), no desired product was formed. So, we
used AB-PCP ester 33 (see the SI) as an alternative highly
potent AB motif.7 Target compounds were purified by reverse-
phase high-performance liquid chromatography (HPLC).
The MIC values, gyrase inhibition, and activity in the
presence of AlbD in an agar diffusion assay were determined
for all six compounds (Table 1).8 All six novel derivatives
maintained their activity in the presence of AlbD (Figure S1,
Supporting Information) and showed overall an improvement in
antibacterial activities when compared with previously reported
isosteres bearing a triazole (34) or a sulfonamide (35) (34, 35:
Table 1 and Figure S3, Supporting Information)6 and also when
compared with the lead compound aza-His albicidin (2). Parent
compound 2 is highly active against G−ve E. coli and S.
typhimurium, whereas isosteres 3, 4, and 8 are equally or up to 4
times less active and isostere 6 is between 2 and 16 times less
active. Interestingly, isosteres 5 and 7 showed an up to four-fold
enhancement (compared with 2) in the activity against G−ve
bacteria. Furthermore, almost all isosteres 3−7 show an
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c02312.
General procedures, characterization of new compounds,
copies of NMR spectra, AlbD cleavage, and MIC assays
(PDF)
AUTHOR INFORMATION
Corresponding Author
■
Roderich D. Sussmuth − Institut fur Chemie, Technische
̈
̈
Universität Berlin, 10623 Berlin, Germany; orcid.org/
0000-0001-7027-2069; Email: Suessmuth@chem.tu-
berlin.de
Authors
Leonardo Kleebauer − Institut fu
Universität Berlin, 10623 Berlin, Germany
̈
r Chemie, Technische
Lieby Zborovsky − Institut fur Chemie, Technische Universität
̈
Berlin, 10623 Berlin, Germany
Kay Hommernick − Institut fu
̈
r Chemie, Technische Universität
Berlin, 10623 Berlin, Germany
D
https://doi.org/10.1021/acs.orglett.1c02312
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Maria Seidel − Institut fu
pubs.acs.org/OrgLett
Letter
(11) Choudhary, A.; Raines, R. T. An Evaluation of Peptide-Bond
Isosteres. ChemBioChem 2011, 12, 1801−1807.
(12) Hanamoto, T.; Kobayashi, T. Cross-Coupling Reactions of (1-
Fluorovinyl)methydiphenylsilane1with Aryl Halides and Aryl Triflates.
J. Org. Chem. 2003, 68, 6354−6359.
̈
r Chemie, Technische Universität
Berlin, 10623 Berlin, Germany
r Chemie, Technische Universität
John B. Weston − Institut fu
Berlin, 10623 Berlin, Germany
̈
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c02312
(13) Sarkar, D.; Melkonyan, F. S.; Gulevich, A. V.; Gevorgyan, V.
Twofold Unsymmetrical C-H Functionalization of PyrDipSi-Substi-
tuted Arenes: A General Method for the Synthesis of Substituted meta-
Halophenols. Angew. Chem., Int. Ed. 2013, 52, 10800−10804.
(14) Belleau, B.; Malek, G. New convenient reagent for peptide
syntheses. J. Am. Chem. Soc. 1968, 90, 1651−1652.
(15) McDermott, J. R.; Benoiton, N. L. N-Methylamino Acids in
Peptide Synthesis. IV. Racemization and Yields in Peptide-bond
Formation. Can. J. Chem. 1973, 51, 2562−2570.
(16) von Eckardstein, L.; Petras, D.; Dang, T.; Cociancich, S.; Sabri,
S.; Grätz, S.; Kerwat, D.; Seidel, M.; Pesic, A.; Dorrestein, P. C.; Royer,
Author Contributions
L.K. designed and synthesized 3−7, analyzed the data, and wrote
the manuscript. L.Z. designed and synthesized 8. M.S. was
responsible for biological testing. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
M.; Weston, J. B.; Sussmuth, R. D. Total Synthesis and Biological
̈
Assessment of Novel Albicidins Discovered by Mass Spectrometric
Networking. Chem. - Eur. J. 2017, 23, 15316−15321.
ACKNOWLEDGMENTS
■
(17) Wipf, P. S.; Erin, M.; Mann, A. Conformational Restriction and
Steric Hindrance in Medicinal Chemistry. The Practice of Medicinal
Chemistry 2015, 279−297.
We acknowledge financial support granted by the Deutsche
Forschungsgemeinschaft (DFG, SU 239/11-1, PE 2600/1-1,
and RTG 2473 “Bioactive Peptides”) (L.K. and K.H.) and the
Alexander-von-Humboldt-Foundation (L.Z.). We also thank
Dr. Andi Mainz (TU Berlin) for valuable discussions, Dr.
Simone Kosol (TU Berlin) for AlbD purification, and Marcello
Spinczyk (TU Berlin) and Marcel Kulike (TU Berlin) for their
contribution in the laboratory. Lastly, we thank Marc Griffel
(TU Berlin) and Arnar Sigurdsson (TU Berlin) for HPLC-MS
measurements.
(18) Meanwell, N. A. Synopsis of Some Recent Tactical Application of
Bioisosteres in Drug Design. J. Med. Chem. 2011, 54, 2529−2591.
(19) Elgaher, W. A. M.; Hamed, M. M.; Baumann, S.; Herrmann, J.;
Siebenbu
Muller, R.; Hartmann, R. W. Cystobactamid 507: Concise Synthesis,
̈
rger, L.; Krull, J.; Cirnski, K.; Kirschning, A.; Brönstrup, M.;
̈
Mode of Action, and Optimization toward More Potent Antibiotics.
Chem. - Eur. J. 2020, 26, 7219−7225.
(20) Huttel, S.; Testolin, G.; Herrmann, J.; Planke, T.; Gille, F.;
̈
Moreno, M.; Stadler, M.; Brönstrup, M.; Kirschning, A.; Muller, R.
̈
Discovery and Total Synthesis of Natural Cystobactamid Derivatives
with Superior Activity against Gram-Negative Pathogens. Angew.
Chem., Int. Ed. 2017, 56, 12760−12764.
REFERENCES
■
(1) Plackett, B. Why big pharma has abandoned antibiotics. Nature
2020, 586, S50−S52.
(21) Testolin, G.; Cirnski, K.; Rox, K.; Prochnow, H.; Fetz, V.;
Grandclaudon, C.; Mollner, T.; Baiyoumy, A.; Ritter, A.; Leitner, C.;
Krull, J.; Van Den Heuvel, J.; Vassort, A.; Sordello, S.; Hamed, M. M.;
(2) Martens, E.; Demain, A. L. The antibiotic resistance crisis, with a
focus on the United States. J. Antibiot. 2017, 70, 520−526.
(3) BIRCH, R. G.; PATIL, S. S. Preliminary Characterization of an
Antibiotic Produced by Xanthomonas albilineans Which Inhibits DNA
Synthesis in Escherichia coli. Microbiology 1985, 131, 1069−1075.
(4) Cociancich, S.; Pesic, A.; Petras, D.; Uhlmann, S.; Kretz, J.;
Schubert, V.; Vieweg, L.; Duplan, S.; Marguerettaz, M.; Noëll, J.;
Elgaher, W. A. M.; Herrmann, J.; Hartmann, R. W.; Muller, R.;
̈
Brönstrup, M. Synthetic studies of cystobactamids as antibiotics and
bacterial imaging carriers lead to compounds with high in vivo efficacy.
Chemical Science 2020, 11, 1316−1334.
(22) Arrata, I.; Grison, C. M.; Coubrough, H. M.; Prabhakaran, P.;
Little, M. A.; Tomlinson, D. C.; Webb, M. E.; Wilson, A. J. Control of
conformation in α-helix mimicking aromatic oligoamide foldamers
through interactions between adjacent side-chains. Org. Biomol. Chem.
2019, 17, 3861−3867.
̈
Pieretti, I.; Hugelland, M.; Kemper, S.; Mainz, A.; Rott, P.; Royer, M.;
Sussmuth, R. D. The gyrase inhibitor albicidin consists of p-
aminobenzoic acids and cyanoalanine. Nat. Chem. Biol. 2015, 11,
195−197.
(5) Kretz, J.; Kerwat, D.; Schubert, V.; Grätz, S.; Pesic, A.; Semsary, S.;
̈
(23) Huc, I. Aromatic Oligoamide Foldamers. Eur. J. Org. Chem. 2004,
2004, 17−29.
(24) Karad, S. N.; Pal, M.; Crowley, R. S.; Prisinzano, T. E.; Altman, R.
A. Synthesis and Opioid Activity of Tyr1-ψ[(Z)CF = CH]-Gly2 and
Tyr1-ψ[(S)/(R)-CF3CH-NH]-Gly2Leu-enkephalin Fluorinated Pep-
tidomimetics. ChemMedChem 2017, 12, 571−576.
Cociancich, S.; Royer, M.; Sussmuth, R. D. Total Synthesis of Albicidin:
̈
A Lead Structure fromXanthomonas albilineansfor Potent Antibacterial
Gyrase Inhibitors. Angew. Chem., Int. Ed. 2015, 54, 1969−1973.
(6) Behroz, I.; Durkin, P.; Gratz, S.; Seidel, M.; Rostock, L.; Spinczyk,
M.; Weston, J. B.; Sussmuth, R. D. Extensive Structure-Activity
Relationship Study of Albicidin’s C-Terminal Dipeptidic p-Amino-
benzoic Acid Moiety. Chem. - Eur. J. 2019, 25, 16538.
(7) Behroz, I.; Kleebauer, L.; Hommernick, K.; Seidel, M.; Grätz, S.;
Mainz, A.; Weston, J. B.; Sussmuth, R. Acetylenic Replacement of
̈
Albicidin’s Methacrylamide Residue Circumvents Detrimental (E)-
(Z)-Photoisomerization and Preserves Antibacterial Activity. Chem. -
Eur. J. 2021, 27, 9077.
(8) Vieweg, L.; Kretz, J.; Pesic, A.; Kerwat, D.; Grätz, S.; Royer, M.;
Cociancich, S.; Mainz, A.; Sussmuth, R. D. The Albicidin Resistance
̈
Factor AlbD Is a Serine Endopeptidase That Hydrolyzes Unusual
Oligoaromatic-Type Peptides. J. Am. Chem. Soc. 2015, 137, 7608−
7611.
(9) Kumari, S.; Carmona, A. V.; Tiwari, A. K.; Trippier, P. C. Amide
Bond Bioisosteres: Strategies, Synthesis, and Successes. J. Med. Chem.
2020, 63, 12290−12358.
(10) Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design
and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018,
61, 5822−5880.
E
https://doi.org/10.1021/acs.orglett.1c02312
Org. Lett. XXXX, XXX, XXX−XXX